The present invention is a method for reproducing coniferous plants by somatic embryogenesis using the techniques of plant tissue culture. It is especially suited for producing large clones of superior trees useful for reforestation.
Loblolly pine (Pinus taeda), its closely related southern pines, and Douglas-fir (Pseudotsuga menziesii) are probably the most important commercial species of temperate North American timber trees. Since the early 1940s, when serious private reforestation efforts began, literally billions of one and two year old nursery-grown trees have been planted on cut-over or burned forest lands. For many years these seedling trees were grown using naturally produced seed from cones collected as a part time effort of individuals seeking to supplement their incomes. As early as 1957 forest geneticists began to plant seed orchards using either seed or grafted scions obtained from superior trees. These trees were selected for such inheritable characteristics as rapid growth, straightness of bole, wood density, etc. Now in both the southern pine and Douglas-fir regions the bulk of the seed is produced from selected trees grown in seed orchards, some of them now second generation orchards.
Despite the fact that the orchards were stocked with superior trees, pollination often cannot be carefully controlled and frequently the seed trees are fertilized by wild pollen of unknown characteristics. For this reason, the characteristics of the progeny produced by sexual reproduction have not been as predictable as hoped and genetic gain could not be attained as rapidly as desired.
Beginning about 1960, techniques were developed for reproducing some species of plants by tissue culture. These were predominately angiosperms and usually ornamental house plants. The method employed use of a suitable explant or donor tissue from a desirable plant. This was placed on a series of culture media in which nutrients and growth hormones were carefully controlled from step to step. The usual progression was growth from the explant to a callus. The callus was placed on a budding medium where adventitious buds formed. These, in turn, were separated, elongated, and rooted to ultimately form plantlets. A plantlet has the nature of a seedling but is genetically identical to the explant donor plant.
Gymnosperms in general, and most forest tree species in particular, proved to be much more difficult to reproduce by tissue culture. It was not until about 1975 that Douglas-fir was successfully reproduced by organogenesis. Loblolly pine was successfully reproduced about two years later.
Culture by organogenesis is tedious and expensive due to the large amount of delicate manual handling necessary. It was soon recognized that embryogenesis was potentially a much more desirable method from the standpoints of quantity of plantlets produced, cost, and potential genetic gain. Work on embryogenesis of forest species began in the late 1970s. U.S. Pat. No. 4,217,730 to El-Nil describes one early process for embryogenesis of Douglas-fir. This approach was later set aside because advanced stage embryos and plantlets could not be readily obtained. However, other workers entered the field in increasing numbers and progress has been rapid even if it has not until the present time reached the commercial stage. A brief review of some of the most important work will follow. This is intended to be representative and is not fully inclusive of all the work in the field. Literature citations in the text are given in abbreviated form. Reference should be made to the bibliography at the end of the specification for full details of the literature cited.
The natural embryogeny of gymnosperms is described in great detail by Singh (1978). Conifer-type embryogeny is one of four types noted for gymnosperms. This includes virtually all of the important forest species except Sequoia.
Bourgkard and Favre (1988) describe what is the apparently successful production of plantlets by somatic embryogenesis of Sequoia sempervirens. As a historic note, this was one of the first forest tree species successfully reproduced by organogenesis.
Hakman and her coworkers have concentrated on Norway spruce (Picea abies), apparently with some success. In a paper by Hakman, Fowke, von Arnold, and Eriksson (1985) the authors describe the production of "embryos" but not plantlets. Hakman and von Arnold (1985) do suggest that they have successfully obtained plantlets. This latter paper is interesting for its comments on the variability within the species and the poor success with many of the seed sources used for explants. The authors suggest that this variability may be due to the physiological condition of the source material. However, other workers have noted great differences in behavior between recognized genotypes of the species.
Nagmani and Bonga (1985) describe embryogenesis from megagametophytes of Larix decidua by tissue culture. Some of the resulting embryos were stated to have further advanced to become plantlets of unknown ploidy established in soil.
Successful production of small quantities of plantlets has now been reported for loblolly pine. Teasdale, Dawson, and Woolhouse (1986) showed the criticality of proper mineral nutrients for cell suspension cultures of loblolly pine. The article by Becwar, Wann, and Nagmani (1988) is enlightening for the differences shown in performance between different families (or genotypes). Three families out of the ten tried accounted for most of their success. Even so, they appeared unable to grow cotyledonary embryos. A companion paper by Nagmani and Becwar (1988) showed development of Pinus taeda to the precotyledonary stage. In an earlier paper, Gupta and Durzan (1987) described their success in taking loblolly pine to the plantlet stage by embryogenesis. However, only one genotype was successfully taken to the plantlet stage and only one plantlet was produced. The authors note the need for "improved conversion rates" as well as other information before the process can be considered commercially practical.
Sugar pine (Pinus lambertiana) has also been cultured to the plantlet stage as reported by Gupta and Durzan (1986). The authors note a very low 1-2% conversion of embryos into plantlets.
The above researchers have also had success in producing Douglas-fir plantlets (Durzan and Gupta 1987). Again, the success ratio appears to be very low and with only a single genotype.
In an earlier application, U.S. Ser. No. 321,035, filed Mar. 9, 1989, now U.S. Pat. No. 4,957,866, the present inventors described an improved method for reproducing coniferous species by somatic embryogenesis. An intermediate high osmoticant culture medium was used to generate strong late stage proembryos, prior to the development of cotyledonary embryos in a medium containing abscisic acid. This application is herein incorporated by reference.
Activated charcoal has been widely used in tissue culture media where it is believed to function as an adsorbent for toxic metabolic products and undesirable amounts of residual hormones. Abscisic acid has also been recognized as being a useful plant hormone in cultures inducing conifer embryogenis; e.g., Boulay, Gupta, Krogstrup, and Durzan (1988). The combination of these two materials has been used by a number of workers, generally with indifferent or negative results. Johansson, Andersson, and Ericksson (1982) cultured anthers of several ornamental plant species using a two phase liquid over solid medium in which the agarified solid phase contained activated charcoal. The charcoal appeared to be useful for absorbing small amounts endogenous abscisic acid. In a related paper Johansson (1983) added exogenous abscisic acid in amounts varying by orders of magnitude from 10.sup.-9 M to 10.sup.-3 M to a charcoal containing medium. His conclusion was that abscisic acid completely inhibits embryogenesis at concentration above 10.sup.-4 M. Further, he noted that the activated charcoal reduced the level of exogenous abscisic acid in the other cultures having lower concentrations thus allowing embryogenesis to proceed.
Ziv and Gadasi (1986) studied embryogenesis in several genotypes of cucumber (Cucumis sativus L.). They used liquid cultures as well as the two layer technique with activated charcoal in the solid layer of the medium and low (0.4 .mu.M) levels of abscisic acid in the liquid layer. In the liquid cultures abscisic acid by itself only slightly improved embryo formation and was significantly more effective than the combination of abscisic acid with activated charcoal. Plantlet development in the liquid over solid cultures was slightly improved by the combination of the two materials.
Buchheim, Colburn, and Ranch (1989) suggest that exogenous abscisic acid and activated charcoal would probably not be a very useful combination of ingredients in a culture medium because of adsorption of the abscisic acid by the charcoal with subsequent loss of its biological effectiveness.
To the present inventor's knowledge, no one has heretofore shown the combination of abscisic acid and activated charcoal to be useful in the embryogenic tissue culture of coniferous species.
While the potential for achieving genetic gain using somatic embryogenesis is recognized as being very great, the problems to date have been so overwhelming that no commercial application has seemed close at hand for forest species. In fact, oil palm is the only tree reproduced by embryogenesis to have reached the stage of small plantation plantings. Until very recent times, possible commercial production of timber species by embryogenesis has remained no more than a fond hope in the minds of the people working in the field.